GSM7135355: HEK293 TDG-N140A anti-MBD3; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
MBD3
Cell type
Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
HEK293
cell line
HEK293
cell type
Human embryonic kidney
treatment
TDG-N140A
chip antibody
MBD3 (abcam, ab157464)
geo_loc_name
missing
collection_date
missing
Sequenced DNA Library
library_name
GSM7135355
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
150mm tissue culture dishes containing 90% confluent cells from each experimental condition were cross-linked and immunoprecipitated with the Active Motif ChIP-IT High Sensitivity Kit according to manufacturers instructions and using indicated antibodies. Samples were sonicated on the Bioruptor (Diagenode) in 1.5 mL Eppendorf tubes at high power for two 10-minute cycles of 30 seconds on and 30 seconds off, replacing warmed water with ice-cold water and minimal ice between each cycle. 25 µL of sonicated chromatin was purified according to ChIP-IT HS Kit protocol and quantified by NanoDrop. 30 µg input chromatin was used for immunoprecipitation. Libraries were generated from 25 µL of fragmented DNA (range 100-300 bp) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), as per the manufacturer's recommendations. Adapters and PCR primers were purchased from Integrated DNA Technologies (IDT). Size selection was carried out using SparQ beads (QIAGEN) prior to PCR amplification (12 cycles). Libraries were quantified using the KAPA Library Quanitification Kits - Complete kit (Universal) (Kapa Biosystems). Average fragment size was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized, pooled, and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 175 pM (HEK293 cells) or 200pM (mouse cortices) on a Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 2x100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 1% level.